Production of adeno‐associated viral vector serotype 6 by triple transfection of suspension HEK293 cells at higher cell densities

Abstract

In recent years, the use of adeno‐associated viruses (AAVs) as vectors for gene and cell therapy has increased, leading to a rise in the amount of AAV vectors required during pre‐clinical and clinical trials. AAV serotype 6 (AAV6) has been found to be efficient in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG), making large‐scale production of AAV6 necessary. Suspension cell‐based platforms are currently limited to low cell density productions due to the widely reported cell density effect (CDE), which results in diminished production at high cell densities and decreased cell‐specific productivity. This limitation hinders the potential of the suspension cell‐based production process to increase yields. In this study, we investigated the improvement of the production of AAV6 at higher cell densities by transiently transfecting HEK293SF cells. The results showed that when the plasmid DNA was provided on a cell basis, the production could be carried out at medium cell density (MCD, 4 × 106 cells mL−1) resulting in titers above 1010 VG mL−1. No detrimental effects on cell‐specific virus yield or cell‐specific functional titer were observed at MCD production. Furthermore, while medium supplementation alleviated the CDE in terms of VG/cell at high cell density (HCD, 10 × 106 cells mL−1) productions, the cell‐specific functional titer was not maintained, and further studies are necessary to understand the observed limitations for AAV production in HCD processes. The MCD production method reported here lays the foundation for large‐scale process operations, potentially solving the current vector shortage in AAV manufacturing.

Enhancing the production of adeno‐associated virus (AAV)2 and AAV9 with high full capsid ratio in HEK293 cells through design‐of‐experiment optimization of triple plasmid ratio

“…Adeno-associated virus (AAV) vectors are increasingly being used in gene therapy because of their safety, diverse tropism, sustained expression, and ability to effectively infect both dividing and nondividing cells. [1][2][3][4][5] To date, six AAV-based gene therapies have been approved for clinical use by regulatory agencies and more than 255 clinical trials have been conducted or are ongoing. [6] To date, more than 100 AAV serotypes, including synthetic serotypes, have been identified that exhibit various tropisms and characteristics in human tissues.…”

Enabling mRNA Therapeutics: Current Landscape and Challenges in Manufacturing

“…Their ability to be manufactured through cell-free systems eliminates the time constraints and possible contaminants associated with traditional viral vector-based gene and cell therapies [10,11]. Therapeutic use of adenoassociated viruses, for example, require high doses that are difficult to accommodate with the current productivity and scalability issues [12], while enzymatic mRNA production is linearly scalable [13], thus achieving the required production more easily. Moreover, a new mRNA product implies a new sequence, but the physicochemical characteristics of an mRNA remain the same, meaning that the manufacturing process can be replicated with minimal changes for a powerful plug-and-play platform [14].…”